Elastic tissue matrix derived hydrogel

ABSTRACT

A tissue-derived hydrogel, as well as methods of making and using such hydrogels, are provided.

This application is a continuation application of U.S. application Ser.No. 14/133,108, filed Dec. 18, 2013; which is a continuation applicationof U.S. application Ser. No. 13/415,355, filed Mar. 8, 2012; whichclaims the benefit under 35 U.S.C. § 119 to U.S. Provisional ApplicationNo. 61/451,315, filed on Mar. 10, 2011. The entire contents of each ofthe aforementioned applications are incorporated herein by reference intheir entirety.

The present disclosure relates generally to tissue-derived hydrogels andmethods of making and using hydrogels for various therapeutic purposes.

Hydrogels have a number of medical and surgical applications, includingtheir use as drug carriers for controlled medicinal delivery, as softtissue fillers, as wound dressings, and as scaffolds for tissuetreatment, regeneration, and/or repair. Existing hydrogels are made fromvarious biomaterials of synthetic and biological origin. However, theextensive chemical modification and synthetic materials required toproduce existing hydrogels result in poor biocompatibility that canhinder tissue treatment or regeneration.

In certain embodiments, a tissue-derived hydrogel is provided. Thehydrogel comprises an acellular arterial tissue matrix that has beentreated with an elastase to form a hydrogel. In further embodiments, amethod is provided for preparing a tissue-derived hydrogel. The methodcomprises harvesting an arterial tissue, decellularizing the tissue, andtreating the arterial tissue with an elastase, thereby causing thetissue matrix to substantially swell and soften. In still furtherembodiments, a hydrogel prepared by any one of the methods disclosed inthe present disclosure is provided. In even further embodiments, amethod of treating a tissue after the removal of bulk soft tissue isprovided, comprising implanting an artery-derived hydrogel into thetissue.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of elastin degradation in porcine carotid artery as afunction of elastase concentration, as described in Example 2. Elastindegradation is indicated by the loss of tissue mass as elastase degradeselastin. The percentage of tissue remaining is calculated as apercentage of weight after digestion compared to pre-digestion weight.

FIG. 2 is a plot of elastin degradation plotted as a function of time,as described in Example 2. The dotted curve represents elastindegradation in tissue that has been decellularized prior to elastindegradation. The solid curve represents elastin degradation in tissuethat has not yet been decellularized when treated with elastase. Elastindegradation is indicated by the loss of tissue mass over time. Thepercentage of tissue remaining is calculated as a percentage of weightafter digestion compared to pre-digestion weight.

FIG. 3 shows examples of porcine aorta treated with elastase, asdescribed in Example 3. At the left is a sample of untreated aorta. Themiddle sample is aorta that has been treated with 1.0 units/ml ofelastase, as described in Example 3. At the right is a sample of aortatreated with 2.0 units/ml of elastase, as described in Example 3.

FIG. 4A-4F shows stained aortic tissue viewed by microscopy, asdescribed in Example 3. FIG. 4A shows Hematoxylin (H&E) staining offresh aorta. FIG. 4B shows H&E staining of decellularized aorta. FIG. 4Cshows H&E staining of elastase-treated aorta. FIG. 4D shows Verhoff'sstaining of fresh aorta. FIG. 4E shows Verhoff's staining ofdecellularized aorta. FIG. 4F shows Verhoff's staining of elastasetreated aorta.

FIG. 5A-5F shows stained aortic tissue viewed by microscopy, asdescribed in Example 3. FIG. 5A shows Alcine blue staining of freshaorta. FIG. 5B shows Alcine blue staining of decellularized aorta. FIG.5C shows Alcine blue staining of elastase-treated aorta. FIG. 5D showsTrichrome staining of fresh aorta. FIG. 5E shows Trichrome staining ofdecellularized aorta. And FIG. 5F shows Trichrome staining ofelastase-treated aorta.

FIG. 6A-6D shows Sirius Red staining of the loosely-packed collagenfiber network of a hydrogel derived from arterial tissue described inExample 3, as viewed at 4× and 10× magnifications. FIG. 6A shows thecollagen network at 4× magnification under bright field. FIG. 6B showsthe same view but under polarized light.

FIG. 6C shows the same bright field view of the hydrogel under 10×magnification. And FIG. 6D shows the same 10× view but under polarizedlight.

FIG. 7 is a scanning electron micrograph image of an artery-derivedhydrogel, as described in Example 3.

FIG. 8 is a graph showing examples of differential scanning calorimetry(DSC) thermograms for fresh porcine aorta and hydrogels, produced asdescribed in Example 3, before and after e-beam sterilization (17.5kGy).

FIG. 9 is a graph showing the softness and flexibility of porcinemuscle, bovine liver, decellularized aorta, and aorta-derived hydrogelas measured by a durometer.

FIG. 10 shows that aorta-derived hydrogels conform to the shapes of thecontainers in which they are placed.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Reference will now be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. Also in this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”are not limiting. Any range described herein will be understood toinclude the endpoints and all values between the endpoints.

As used herein, “hydrogel” means any soft biomaterial composed ofpolymer. Hydrogels may be created synthetically using artificialmaterials, or they may be produced by processing natural tissues. Asused herein, “native” refers to the cells, tissues, or organs present inan animal prior to hydrogel implantation, or to the tissue used to forma hydrogel prior to any processing to degrade elastin or decellularizethe tissue.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

The present disclosure relates to hydrogels derived from acellulartissue matrices. These hydrogels can be implanted in or grafted onto asubject such as a vertebrate subject. The present disclosure alsoprovides methods of producing tissue-derived hydrogels.

In certain embodiments, a hydrogel matrix is derived from a vertebratearterial tissue. In some embodiments, the vertebrate arterial tissue isextracted from pig. In other embodiments, species that can serve asarterial tissue donors include, without limitation, human, non-humanprimates (e.g. monkeys, baboons, or chimpanzees), pig, cow, horse, goat,sheep, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, or mouse. Incertain embodiments, the animal that serves as an arterial tissue donorcan be a transgenic animal. In further embodiments, the transgenicanimal lacks expression of certain antigens, thereby increasing hydrogeltolerance after implant. In certain embodiments, the host animal tissueis altered to lack α-galactose (α-gal).

Elimination of the α-gal epitopes from the collagen-containing arterialmaterial may diminish the immune response against the arterial material.The α-gal epitope is expressed in non-primate mammals and in New Worldmonkeys (monkeys of South America). U. Galili et al., J. Biol. Chem.263: 17755 (1988). This epitope is absent in Old World primates (monkeysof Asia and Africa and apes) and humans, however. Id. Anti-Galantibodies are produced in humans and primates as a result of an immuneresponse to the α-gal epitope carbohydrate structures ongastrointestinal bacteria. U. Galili et al., Infect. Immun. 56: 1730(1988); R. M. Hamadeh et al., J. Clin. Invest. 89: 1223 (1992).

Since non-primate mammals (e.g., pigs) produce α-gal epitopes,xenotransplantation of arterial material from these mammals intoprimates can result in rejection because of primate anti-Gal binding tothe α-gal epitopes on the arterial material. The binding results in thedestruction of the arterial material by complement fixation and byantibody dependent cell cytotoxicity. U. Galili et al., Immunology Today14: 480 (1993); M. Sandrin et al., Proc. Natl. Acad. Sci. USA 90: 11391(1993); H. Good et al., Transplant. Proc. 24: 559 (1992); B. H. Collinset al., J. Immunol. 154: 5500 (1995). Furthermore, xenotransplantationresults in major activation of the immune system to produce increasedamounts of high affinity anti-Gal antibodies.

In some embodiments, when animals that produce α-gal epitopes are usedas the hydrogel tissue source, the animals are genetically engineeredusing methods known in the art to substantially eliminate α-galexpression. In other embodiments, α-gal is removed from harvested tissueusing enzymatic processing. Removal of α-gal from host tissue can reducethe immune response that is associated with anti-Gal antibody binding toα-gal epitopes after a hydrogel is implanted in a tissue.

To enzymatically remove α-gal epitopes, after washing the tissuethoroughly with saline, the tissue sample may be subjected to one ormore enzymatic treatments to remove certain immunogenic antigens, ifpresent in the sample. In some embodiments, the tissue sample may betreated with an α-galactosidase enzyme to eliminate α-gal epitopes, ifpresent in the tissue. In some embodiments, the tissue sample is treatedwith α-galactosidase at a concentration of 300 U/L prepared in 100 mMphosphate buffered saline at pH 6.0. In other embodiments, theconcentration of α-galactosidase is increased to 325, 350, 375, 400,425, 450, 475, or 500 U/L, or reduced to 275, 250, 225, or 200 U/L (orany concentration in between). In other embodiments, any suitable enzymeconcentration and buffer can be used as long as sufficient antigenremoval is achieved.

Alternatively, in certain embodiments animals that have been geneticallymodified to lack one or more antigenic epitopes may be selected as thehydrogel tissue source. For example, animals (e.g., pigs) that have beengenetically engineered to lack the terminal α-galactose moiety can beselected as the tissue source. For descriptions of appropriate animalssee U.S. application Ser. No. 10/896,594 and U.S. Pat. No. 6,166,288,the disclosures of which are incorporated herein by reference in theirentirety. In addition, certain exemplary methods of processing tissuesto reduce or remove alpha-1,3-galactose moieties are described in Xu, etal., “A Porcine-Derived Acellular Dermal Scaffold that Supports SoftTissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose andRetention of Matrix Structure,” Tissue Engineering, Vol. 15, 1-13(2009), which is incorporated by reference in its entirety.

In various embodiments, arterial tissue is harvested from a donorvertebrate and cleaned to remove blood. In some embodiments, thearterial tissue is first subjected to multiple rounds of freeze/thaw todisrupt the tissue. In certain embodiments, the harvested arterialtissue is then decellularized and treated with elastase to disrupt theelastin network. In some embodiments, after decellularization andelastase treatment, the arterial tissue is washed.

In various embodiments, natural hydrogel matrices derived fromelastase-treated arterial tissue exhibit higher biocompatibility overartificially produced hydrogels. In certain embodiments, hydrogelmatrices derived from elastase-treated arterial tissue have reducedimmunogenicity compared to synthetic hydrogels when implanted in atissue in need of treatment.

In certain embodiments, elastase-treated arterial tissue is flexible andtransparent, with a consistency similar to that of silicone gel orputty. In other embodiments, elastase-treated arterial tissue swells andbecomes more malleable than un-treated arterial tissue. The relaxationof the extracellular matrix caused by elastin degradation allows thetissue to absorb significant amounts of water, causing the hydrogel toswell and obtain the consistency of native soft tissue. The relaxationalso increases the malleability of the hydrogel, enabling it to bemolded into desired forms or structures. At the same time,elastase-treated arterial tissue retains high levels of proteoglycansand other signaling molecules that can enhance biocompatibility andpromote native tissue re-growth when implanted in a tissue in need oftreatment. Further, elastase-treated arterial tissue retains a strongcollagen network that preserves structural integrity. In furtherembodiments, hydrogels produced according to the methods described aboveretain some of their elastin network (i.e., the elastin network is onlypartially degraded by elastase) and thus retain a higher level ofrigidity. In various embodiments, partially elastin-digested hydrogelscan be used as tissue fillers where more rigid tissue fillers arerequired.

In certain embodiments, the malleability of elastase-treated arterialtissue provides for several beneficial properties. In furtherembodiments, the degraded arterial tissue can provide the physicalproperties normally associated with a synthetic hydrogel. Thus, thedegraded arterial tissue can be molded into various shapes and used as asoft tissue implant following surgical removal of a soft tissue. Infurther embodiments, degraded arterial tissue retains structuralintegrity through the remaining collagen network. Thus, in someembodiments, rather than dissolving, dissociating, tearing, ordeforming, molded arterial tissue hydrogels can retain their shape andstructural integrity after implant into a soft tissue.

In various embodiments, a hydrogel matrix can be produced bydecellularizing arterial tissue followed by elastase treatment. Incontrast, dermis is not effective for hydrogel preparation, regardlessof the elastase concentration or elastase exposure time. In furtherembodiments, the arterial tissue can be aortic tissue. The aorta is thelargest arterial tissue region and thus can provide the most arterialtissue mass for hydrogel production. In certain embodiments, the aortais harvested from a pig.

In various embodiments, the harvested arterial tissue is cleaned toremove blood. In some embodiments, the harvested tissue is subjected tomultiple rounds of freeze/thaw to disrupt the tissue. During the freezeportion of the freeze/thaw procedure, ice crystals form, which expandand disrupt the tissue. During the thaw portion, holes or cracks arecreated in the tissue, which subsequently allow enzymes such as elastaseto penetrate more quickly and deeply into the tissue. In someembodiments, the arterial tissue is subjected to 1, 2, 3, 4, or 5 roundsof freeze/thaw to disrupt the tissue.

In certain embodiments, harvested and cleaned arterial tissue isdecellularized in a non protein-denaturing detergent solution. Infurther embodiments, the decellularized arterial tissue is treated withelastase to degrade elastin. In still further embodiments, afterelastase treatment, the arterial tissue matrix is washed in isotonicsolution.

In other embodiments, a hydrogel matrix is derived by first treatingharvested arterial tissue with elastase and then decellularizing. Asused herein, “harvested” arterial tissue is any portion or completearterial tissue that has been separated from its native environment,e.g., by dissection. In certain embodiments, arterial tissue isharvested and cleaned to remove blood. In further embodiments, theharvested tissue is then treated with elastase. In still furtherembodiments, after elastase treatment, the tissue matrix isdecellularized using a non protein-denaturing detergent solution. Thedecellularized tissue matrix is then washed in isotonic solution.

In further embodiments, treatment of arterial tissue with elastase and adecellularizing detergent produces a soft bulk tissue matrix. In furtherembodiments, the soft bulk tissue matrix has similar properties to ahydrogel. In still further embodiments, the soft bulk tissue matrix alsominimizes undesirable crosslinking and retains proteoglycans and othersignaling molecules that promote biocompatibility. In furtherembodiments, the soft bulk tissue matrix demonstrates increasedbiocompatibility due to the use of natural tissue materials and the highlevels of glycosamino-glycans and other growth factors present in thehydrogel matrix. In further embodiments, the soft bulk tissue matrix isuseful as a tissue filler for tissue regeneration after the loss of bulksoft tissue.

In various embodiments, a method is provided for preparing atissue-derived hydrogel. In certain embodiments, the tissue to beharvested is aorta. In further embodiments, the tissue is porcine aorta.In various embodiments, the harvested tissue is first subjected tomultiple rounds of freeze/thaw to break up the tissue. The harvestedtissue is then rinsed with saline. In some embodiments, the saline is a0.9% saline solution. In other embodiments, the saline concentration isreduced to 0.5%, 0.6%, 0.7%, or 0.8%, or is increased to 1.0%, 1.1%,1.2%, 1.3%, 1.4%, or 1.5% saline solution. The harvested tissue is thenwashed in a buffer solution. In certain embodiments, the buffer solutionis Tris-HCL. In further embodiments, the Tris-HCL is at a concentrationof between 30 mM and 120 mM (i.e., at 30 mM, 40 mM, 50 mM, 60 mM, 70 mM,80 mM, 90 mM, 100 mM, 110 mM, or 120 mM, or any concentration inbetween). In still further embodiments, the buffer solution alsocontains at least one antibiotic. In even further embodiments, thebuffer contains 1.5 μg/ml of amphotericin, 65 μg/ml of streptomycin and65 units/ml of penicillin. In other embodiments, the concentration ofamphotericin is decreased to 1.0, 1.1, 1.2, 1.3, or 1.4 μg/ml, orincreased to 1.6, 1.7, 1.8, 1.9, or 2.0 μg/ml (or any concentration inbetween). In certain embodiments, the concentration of streptomycin isdecreased to 60, 61, 62, 63, or 64 μg/ml, or increased to 66, 67, 68,69, or 70 μg/ml (or any concentration in between). In some embodiments,the concentration of penicillin is decreased to 60, 61, 62, 63, or 64μg/ml, or increased to 66, 67, 68, 69, or 70 μg/ml (or any concentrationin between). In further embodiments, the harvested tissue is washed inTris-HCL for 10 minutes. In other embodiments, the harvested tissue iswashed in Tris-HCL for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 45, or 60 minutes (or any time in between).

In various embodiments, the harvested tissue is then treated withelastase. In certain embodiments, between 0.021 and 5.35 units/ml ofelastase are added to the Tris buffer to digest elastin. See FIG. 1.Increasing the elastase concentration does not necessarily result in afaster or more complete elastin degradation. An elastase enzyme unit isdefined as the amount of enzyme that will hydrolyze 1.0 μmole ofN-succinyl-L-Ala-Ala-Ala-p-nitroanilide per min at pH 8.0 and at 25° C.In further embodiments, between 0.1 and 5 units/ml of elastase areadded. In still further embodiments, 0.3 units/ml of elastase is added.In even further embodiments, 0.5 units/ml of elastase is added. Infurther embodiments, 1 unit/ml of elastase is added. In yet anotherembodiment, 2 units/ml of elastase is added. In still furtherembodiments, 0.025, 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3 or 5.35 units/ml of elastase, or any concentration inbetween, are added.

The tissue to be digested is incubated with elastase for between 10 and96 hours, depending on the concentration of elastase and the desiredpercentage of total elastin degradation. In certain embodiments, theelastase incubation time or is increased in order to more fully degradeelastin in the tissue. In other embodiments, the elastase incubationtime is reduced in order to preserve more elastin in the tissuefollowing digestion. In some embodiments, the tissue to be digested isincubated with elastase for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,24, 30, 36, 48, 60, 72, 84, or 96 hours (or any time in between). In oneembodiment, porcine aorta tissue is incubated with elastase for 18hours. In certain embodiments, the tissue is agitated during elastasetreatment. Such agitation may be gentle or more intense. In furtherembodiments, the agitation involves shaking the tissue. In someembodiments, elastase-digested tissue is then washed in saline solution.In certain embodiments, washing is conducted for 30 minutes. In otherembodiments, washing is conducted for 10, 20, 30, 40, 50, 60, 90, or 120minutes.

In various embodiments, the elastase-digested tissue is thendecellularized using a non protein-denaturing detergent solution. Incertain embodiments, the detergent that is selected does not disrupt thestructural integrity or alter the functional properties of the tissuematrix during decellularization. In some embodiments, the detergent issodium deoxycholate (SDC). In other embodiments, the detergent is sodiumdodecyl sulfate (SDS), Triton-X 100 or a combination of SDC, SDS, and/orTriton-X 100. In other embodiments, any known decellularizationdetergent can be used. In certain embodiments, the concentration ofdetergent is calibrated using techniques known to one of skill in theart to ensure that the tissue is completely decellularized. In evenfurther embodiments, 2% sodium deoxycholate is used.

In some embodiments, the detergent solution is applied for at least 24hours (e.g., at least 24, 25, 26, 27, 28, 29, 30, 36, 48, 60, or 72hours, or any time in between). In certain embodiments, the detergentsolution is applied for at least 60 hours (e.g., at least 60, 65, 70,75, 80, 85, 90, or 120 hours, or any time in between). In furtherembodiments, a 2% sodium deoxycholate solution is applied for 64 hoursat 4 degrees Celsius to decellularize harvested porcine aortic tissue.In various embodiments, the decellularized tissue is then washed toremove detergent and lysed cells. In certain embodiments, the washsolution is saline or any saline-containing solution. In otherembodiments, the wash solution is phosphate buffered saline (PBS) andethylenediaminetetraacetic acid (EDTA). In some embodiments, the washsolution is PBS at pH 7.4 and 5 mM EDTA. In further embodiments, thedecellularized tissue is washed for at least one hour (e.g., at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, or 48 hours, or any timein between). In even further embodiments, decellularized tissue iswashed for at least 10 hours. In even further embodiments, the washingis done at nearly room temperature or at any temperature between andincluding room temperature and 4 degrees Celsius (e.g., at a temperatureof 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 18, 15, 10, 5, 4, 3, 2,or 1 degree Celsius, or any temperature in between).

In various embodiments, hydrogels produced by the above methods cansubsequently be cryopreserved. In certain embodiments, the hydrogel isincubated in a cryopreservation solution. In further embodiments, thecryopreservation solution includes one or more cryoprotectants tominimize ice crystal damage to the hydrogel's structural matrix. Incertain embodiments, the hydrogel can be cryopreserved by placing it inthe cryopreservation solution and then freezing it in a freezer atapproximately −80 degrees Celsius (e.g., at −75, −80, or −85 degreesCelsius, or any temperature in between), or by plunging the hydrogelinto liquid nitrogen and storing frozen until use.

In various embodiments, hydrogels are cryopreserved for storage byfreeze-drying. In certain embodiments, the hydrogel is placed in acryopreservation solution that includes components, such as an organicsolvent or water, to protect against damage during freeze drying. Infurther embodiments, following incubation in the cryopreservationsolution, the hydrogel is placed in a sterile vessel that is permeableto water vapor but impermeable to bacteria. The vessel is cooled to alow temperature at a specified rate that is compatible with the specificcryoprotectant formulation to minimize the freezing damage. The hydrogelis then dried at a low temperature under vacuum conditions. At thecompletion of the drying, the vacuum of the freeze drying apparatus isreversed with a dry inert gas such as nitrogen, helium or argon. Thehydrogel is then sealed in an impervious container and stored until use.While the example above describes one method for cryopreservation, oneof skill will recognize that other such methods known in the art may beused to cryopreserve and store hydrogels.

In certain embodiments, artery-derived hydrogels produced according tothe methods described above retain some of the structural and functionalaspects found in untreated tissue. In various embodiments, thebiological functions retained include the ability to support nativetissue spreading, native cell proliferation, and native celldifferentiation. In certain embodiments, the hydrogel retains growthfactors (such as type I collagen, glycosaminoglycans, or proteoglycans)that serve to promote tissue and cell regeneration or growth. In variousother embodiments, the retained physical properties of hydrogels (suchas the maintenance of the three dimensional collagen network and itsstrength, ductility and elasticity) enhance cell growth and tissuespreading. In certain embodiments, the efficiency of the biologicalfunction of a tissue-derived hydrogel can be measured by the ability ofthe hydrogel to support cell proliferation. In further embodiments, thehydrogel is able to promote at least 75%, 50%, 30%, 25%, or 10% (or anypercentage in between) of the proliferation that would occur on a nativetissue or organ scaffold.

While a hydrogel to be implanted in a vertebrate can be produced fromthe same species and the same organ as the host, this is not arequirement. In various embodiments, the hydrogel should retainbiocompatibility and amenability to cell proliferation and tissue growthwhen implanted in a host tissue. Thus, in certain embodiments, ahydrogel derived from the tissue of one species can be implanted inanother species. Exemplary species that can serve as hydrogel tissuedonors include, without limitation, human, non-human primates (e.g.monkeys, baboons, or chimpanzees), pig, cow, horse, goat, sheep, dog,cat, rabbit, guinea pig, gerbil, hamster, rat, or mouse. Likewise, incertain embodiments, a hydrogel derived from one tissue source can beimplanted into a different host tissue. In further embodiments,hydrogels derived from porcine artery (such as aorta) are implanted invarious human tissues.

In various embodiments, artery-derived hydrogels can be provided invariety of forms and sizes depending on the tissue or organ into whichit will be implanted. Thus, in certain embodiments, the hydrogel can beprovide in strips or sheets. In other embodiments, the hydrogel isprovided in unmolded balls or cylinders that can later be manipulated toassume various structures. In even further embodiments, hydrogels areprovided pre-formed to achieve a desired structure for a given tissueimplant use. In other embodiments, an artery-derived hydrogel isprovided as a moldable putty that will conform to the shape of the spacein which it is implanted. For example, the moldable hydrogel canconform, after implantation, to the shape of a void space in a recipienttissue caused by surgical removal of tissue (e.g., tumor removal).

In some embodiments, the artery-derived hydrogel comprises a loosecollagen network that is rich in glycosamino-glycans and other growthfactors. In further embodiments, the elastin in an artery-derivedhydrogel tissue is partially or completely degraded. See FIG. 2.

Unlike dermal or other tissues, arterial tissue contains a high densityof elastin. In some instances, 40 to 50% of the arterial tissue iscomposed of elastin, allowing the native artery to swell and contractwith blood flow. Thus, in some embodiments, elastin degradation disruptsa significant component of the arterial tissue and causes significantswelling and tissue softening that is not observed when other tissuesare degraded with elastase, preventing their use as hydrogels. Infurther embodiments, swelling is due to increased tissue matrixhydration. In certain embodiments, elastase-treated tissue matricesswell by 200 to 300% as compared to undigested arterial tissue whenplaced in aqueous solution (e.g., elastase-treated tissue matrices swellby 200, 225, 250, 275, or 300%, or any percentage in between). Infurther embodiments, hydrogels produced from porcine aortic tissue swellby an average of 278% after elastase treatment, decellularization andplacement in an aqueous solution. In further embodiments, the swellingof arterial tissue induced by elastase treatment produces a soft,putty-like foam.

In certain embodiments, artery-derived hydrogels are molded into variousshapes and demonstrate a good ability to retain that structure overtime. In further embodiments, the artery-derived hydrogels retainstructural integrity (i.e., the hydrogel remains in one piece) aftermechanical manipulation. In even further embodiments, aorta-derivedhydrogels retain structural integrity after application of tensile ortorsional forces.

In various embodiments, artery-derived hydrogels can be used as tissuefillers for tissue repair or treatment. In certain embodiments,artery-derived hydrogels are used as implants for the face or neck. Infurther embodiments, artery-derived hydrogels are used as tissue fillersfor tissue regeneration after the loss of bulk soft tissue. In certainembodiments, artery-derived hydrogels are implanted into a tissue afterthe loss of bulk soft tissue and swell to fill the region of losttissue. In even further embodiments, artery-derived hydrogels are usedas tissue fillers after lumpectomies. In still further embodiments,artery-derived hydrogels are implanted after a lumpectomy and serve tocoagulate blood from the operation site, reducing the need for a drain.

It has been shown that after tumor removal, tissue re-growth is poor,especially as to the subcutaneous tissue layers. Generally, a layer ofskin will regrow after tumor removal, but the underlying tissue remainsunregenerated. Thus, in various embodiments, artery-derived hydrogelscan be used as implants for the face or neck after tumor removal. Incertain embodiments, such implants serve as tissue fillers that canprovide the face or neck with a more natural look after tumor removal.In further embodiments, such implants serve as scaffolds for tissueregeneration and/or repair by providing a structural matrix for nativecell migration and/or proliferation. In still further embodiments, suchimplants help coagulate blood at the site of tumor removal and reducethe need for a drain. In even further embodiments, such implants promotetissue repair or regeneration due to the high levels ofglycosamino-glycans and other growth factors retained in the hydrogel.

In various embodiments, artery-derived hydrogels can be used as deliveryvehicles for pharmaceutical agents. In certain embodiments,artery-derived hydrogels are impregnated with a pharmaceutical agentusing techniques known to one of skill in the art. In furtherembodiments, impregnated hydrogels are then implanted in a tissue inneed of the pharmaceutical agent. In even further embodiments, implantedhydrogels containing pharmaceutical agents can serve as time-releasecarriers for a pharmaceutical agent, releasing the pharmaceutical agentas the implanted hydrogel is gradually dissolved and reabsorbed into thetissue. In various embodiments, the pharmaceutical agent is ananticancer agent such as paclitaxel, 5-fluorouracil, bleomycin A2 andmitomycin C, methotrexate, or doxorubicin. In further embodiments, thepharmaceutical agent is a growth factor such as fibroblast growthfactor, transforming growth factor, bone morphogenetic protein, vascularendothelial growth factor, nerve growth factor, or insulin-like growthfactor. In still further embodiments, the pharmaceutical agent is a painrelief agent such as oxycodone HCl, morphine sulfate, or tramadol. Andin yet further embodiments, the pharmaceutical agent is an antimicrobialagent such as chlorhexidine, ciprofloxacin, clarithromycin,chloramphenicol, ceftriaxone, ofloxacin, polymycin B, sulfamethoxazole,streptomycin, tobramycin, tetracycline, or trimethoprim. In someembodiments, the hydrogel is impregnated with combinations of at leasttwo pharmaceutical agents (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, or 10agents). In certain embodiments, the hydrogel containing thepharmaceutical agent is implanted into a tissue after the removal ofbulk soft tissue. In further embodiments, the hydrogel containing thepharmaceutical agent is implanted in a tissue in need of cancertreatment or therapy. In some embodiments, the hydrogel containing thepharmaceutical agent is implanted in a chronic wound site.

The following examples serve to illustrate, and in no way limit, thepresent disclosure.

EXAMPLES Example 1—Decellularization of Porcine Arteries and Aorta

Porcine carotid arteries (about 10 to 20 cm long) or aorta wereharvested by manual dissection. Blood clots in arteries and aorta werewashed off before decellularization. Carotid arteries and aorta weredecellularized at room temperature (22 to 25° C.) for 24 hours in a 10mM HEPES buffer solution (pH 8.0) containing 1% (w/v) Triton X-100 and10 mM EDTA with gentle agitation on a shaker. Decellularized arteriesand aorta were washed with 0.9% saline to remove the detergent (i.e.,Triton x-100) until foam was no longer observed. Arteries and aorta werethen treated at room temperature (22 to 25° C.) for 24 hours in secondHEPES buffer solution (10 mM, pH 7.4) containing 30 units/ml DNase, 50μg/ml gentamicin, 2 mM calcium chloride and 2 mM magnesium chloride. TheDNase solution was discarded, and tissue was washed three times with0.9% saline (10 min each time). Histological evaluation (H&E stain) andbiochemical tests demonstrated that the process completelydecellularized harvested porcine arteries and aorta. In some cases,decellularized arteries and aorta were further treated inphosphate-buffered saline (pH 6.5) containing 0.2 unit/mlα-galactosidase and 50 mM ETDA. This step eliminated extracellular α-galepitopes of porcine tissue.

Example 2—Removal of Elastin from Porcine Carotid Arteries

This experiment was intended to identify the effective range of elastaseconcentration and time course of elastin removal from porcine carotidarteries and aorta. Fresh porcine carotid arteries were harvested bymanual dissection. Arteries were washed in saline to clean blood clots,and cut into small pieces (1 mm×1 mm). Samples of 80 mg tissue inmicrotubes were treated in 0.5 ml Tris-HCl buffer (100 mM, pH 8.0)containing elastase of between 0.011 and 5.35 units per ml for 22 hours.After incubation, samples were centrifuged, pellets of samples werewashed with de-ionized water, and centrifuged again. Sample pellets werethen lyophilized, and the percentage of tissue remaining after elastasetreatment was calculated. FIG. 1 demonstrated that elastase atconcentration from 0.021 unit/ml to 5.35 units per ml was effective indigesting elastin in porcine arteries. On average, porcine carotidarteries contained about 50% (w/w) elastin. When 5.35 units/ml elastasewas used, the elastin content of porcine carotid artery was reduced by79±14% after 22 hour digestion.

A comparison was made between freshly harvested carotid arteries anddecellularized arteries. Both fresh and decellularized arteries were cutinto small pieces (1 mm×1 mm). 80 mg tissue samples in microtubes weretreated in 0.5 ml Tris-HCl buffer (100 mM, pH 8.0) containing 0.67unit/ml elastase for 5, 8, 14, and 18 hours. At the end of eachincubation point, samples were centrifuged, pellets of samples werewashed with de-ionized water, centrifuged again. Sample pellets werelyophilized, and the percentage of tissue remaining after elastasetreatment was calculated. As shown in FIG. 2, fresh arteries anddecellularized arteries had comparable reaction curves. At 0.67 unit/mlelastase, the reaction was complete after 18 hours.

Example 3—Removal of Elastin from Porcine Aorta

Decellularized aorta (produced as described in Example 1) was washedwith 100 mM Tris-HCl buffer (pH 8.0) for 10 minutes. 2-gram tissuesamples (wet weight) were treated at room temperature for 18 hours in 20ml of 100 mM Tris-HCl buffer (pH 8.0) containing 1.0 unit/ml or 2unit/ml elastase. Tissue material was washed in 0.9% saline for 30 min.FIG. 3 shows the gross appearance of elastase-treated aorta sections.After elastase treatment and washing, the aorta tissue became astructured, soft hydrogel.

In a second experiment, histological evaluation was used to comparefresh, decellularized, and elastase-treated aorta tissues.Decellularization of aorta tissue was done at room temperature for 5hours in a 10 mM HEPES buffer solution (pH 8.0) containing 1% (w/v)Triton x-100 and 10 mM EDTA. Elastase treatment was done for 49 hours in100 mM Tris-HCl buffer (pH 8.0) containing 0.5 unit/ml elastase. Thetissue to solution ratio was 10 ml solution per 1 gram wet aorta tissue.Decellularized and elastase-treated tissue samples were washed in 10 mMHEPES buffer solution. Samples were processed, and stained forhistological evaluation (H&E, Verhoff's, Alcine blue and Trichromestains). FIG. 4 and FIG. 5 show the characteristic histologicalstructures of fresh aorta tissues, and the retention of the tissue'sstructural integrity after decellularization. However, elastasetreatment resulted in tissue swelling and substantial change in thetissue structures (i.e., loosening).

In a third experiment, harvested fresh porcine aorta was rinsed with0.9% saline, and cut open for elastase treatment. Aortic tissue (14grams) was treated at room temperature for 72 hours in 150 ml Tris-HClbuffer (50 mM, pH 8.0) containing 0.3 unit/ml elastase, 65 units/mlpenicillin, 1.5 μg/ml amphotericin and 65 μg/ml streptomycin. Afterelastase treatment, tissue material was washed for 2 hours in a 2% (w/v)sodium deoxycholate detergent solution. The solution of 2% sodiumdeoxycholate was then refreshed, and aorta tissue material was washed at4° C. for an additional 62 hours. In order to remove sodiumdeoxycholate, aorta tissue material was washed three times (10 hourseach) in phosphate-buffered saline (pH 7.4) containing 5 mM EDTA. Afterprocessing, the tissue was weighed to be 53 grams, and had increased involume by 278% as compared to the pre-treatment volume. FIG. 6 shows thenetwork of loosely-packed collagen fibers in an aorta-derived hydrogel.The translucent, thin slices (˜2 mm thick) of aorta-derived hydrogelshown in FIG. 6 were stained with picosirius red, and photos were takenunder bright field and polarized light. A loosely-packed collagen fibernetwork that supports biochemical components and structural elements isvisible in FIG. 6. FIG. 7 shows the fine ultrastructure of anaorta-derived hydrogel material. Differential scanning calorimetry wasalso used to evaluate the change in stability of aorta tissue afterhydrogel processing (FIG. 8). The elastase treatment alters thethermogram data plots significantly. The onset denaturation temperaturehas decreased and denaturation enthalpy increased. Decellularizationalone does not change these two parameters. The decrease of the onsetdenaturation temperature is related to the enhanced hydration of thetissue after elastase treatment, and the enthalpy change could beattributed to the loss of elastin. However, with a denaturationtemperature of less than 50° C. after e-beam sterilization, the hydrogelmaterial is sufficiently stable at body temperature.

Example 4—Biomechanical Properties of Aorta-Derived Hydrogels

The softness and flexibility of aorta-derived hydrogels (N=4) wasanalyzed by a durometer. A durometer measures the indentation resistanceof elastomeric or soft materials based on the depth of penetration of aconical indentor. Hardness values range from 0 to 100. A lower durometerreading indicates a softer material, whereas a higher number indicatesthat the material is harder. Durometer measurements were taken in fivespots for each hydrogel sample tested. Porcine muscle, bovine liver anddecellularized aorta were tested at the same time for comparison. Meandurometer values for porcine muscle, bovine liver and decellularizedaorta were 29.4±4.9, 7.2±1.4, and 30.5±10.0, respectively (mean±SD;N=4). Durometer readings were zero for all aorta-derived hydrogelsamples (FIG. 9). Aorta-derived hydrogels are soft and flexible enoughthat when multiple pieces were placed together in a container, theyconformed to the shape of the container, produce a flat surface, andleft no empty space between the pieces (FIG. 10).

While the above examples involved the production, structure, and use ofporcine aorta and carotid artery in hydrogels, one of skill wouldrecognize that other tissues could be used to produce tissue-derivedhydrogels having desired properties.

Other embodiments of the disclosed devices and methods will be apparentto those skilled in the art from consideration of the specification andpractice of the devices and methods disclosed herein.

1. A tissue filler comprising: an arterial tissue matrix that has been treated with elastase to produce a swollen and softened arterial tissue matrix having a disrupted elastin network and in the form of a moldable putty that will conform to the shape of a space in which it is implanted, and wherein the arterial tissue matrix has an increased malleability after the elastase treatment as compared to the acellular tissue matrix prior to elastase treatment.
 2. The tissue filler of claim 1, wherein the arterial tissue matrix is aortic tissue.
 3. The tissue filler of claim 2, wherein the aortic tissue is porcine aortic tissue.
 4. The tissue filler of claim 1, wherein the arterial tissue matrix is acellular.
 5. The tissue filler of claim 1, wherein the elastase-treated arterial tissue matrix expands to a volume that is 200-300% larger than the volume of untreated arterial tissue when placed in an aqueous solution.
 6. The tissue filler of claim 4, wherein the arterial tissue matrix can expand after implantation into a tissue to fill a space left by a tissue removal operation.
 7. The tissue filler of claim 1, wherein the arterial tissue matrix is treated with elastase at a concentration of between 0.021 and 5.35 units per ml for 5 to 96 hours.
 8. A method of treating a tissue of the face or neck, comprising: implanting a tissue filler into the tissue, wherein the tissue filler comprises an arterial tissue matrix from which some but not all of the elastin has been removed, and wherein the arterial tissue matrix used to prepare the tissue filler has been subjected to at least one round of freezing and thawing followed by removal of at least some elastin from the arterial tissue matrix, and wherein the arterial tissue matrix is not cross-linked prior to implantation.
 9. The method of claim 8, wherein the treatment with elastase causes the arterial tissue matrix to substantially swell and soften.
 10. The method of claim 8, wherein the arterial tissue matrix is aortic tissue.
 11. The method of claim 10, wherein the aortic tissue is porcine aortic tissue.
 12. The method of claim 10, wherein the arterial tissue matrix is decellularized.
 13. The method of claim 12, wherein a non-denaturing detergent solution is used to decellularize the arterial tissue matrix.
 14. The method of claim 12, wherein decellularizing the arterial tissue matrix includes placing the arterial tissue matrix in a composition containing detergent for at least 24 hours.
 15. The method of claim 8, wherein the elastin has been removed from the arterial tissue matrix by exposing the arterial tissue matrix to elastase at a concentration of between about 0.021 and 5.35 units/ml for 5 to 96 hours.
 16. The method of claim 15, wherein the arterial tissue matrix is placed in the solution containing elastase for 5 to 14 hours in order to partially degrade elastin.
 17. The method of claim 8, wherein the tissue filler swells to fill a space in the tissue of the face or neck.
 18. The method of claim 8, wherein the tissue filler is implanted after removal of a tumor from the face or neck.
 19. The method of claim 18, wherein the tissue filler enhances the coagulation of blood at the site of the tumor removal.
 20. The method of claim 8, wherein the tissue filler promotes tissue repair and regeneration.
 21. The method of claim 8, wherein the tissue filler is impregnated with a pharmaceutical agent and releases the pharmaceutical agent over time as the implanted tissue filler is gradually dissolved and reabsorbed into the tissue.
 22. The method of claim 21, wherein the pharmaceutical agent is an anticancer agent, a growth factor, a pain relief agent, an antimicrobial agent, or combinations thereof. 